Participation of cofilin in opsonized zymosan-triggered activation of neutrophil-like HL-60 cells through rapid dephosphorylation and translocation to plasma membranes

We studied the roles of cofilin, an actin-binding phosphoprotein, in superoxide production of neutrophil-like HL-60 cells triggered by opsonized zymosan (OZ), OZ caused dephosphorylation of cofilin as well as a transient increase of F-actin, Both reactions were complete within 30 s. Okadaic acid (OA) magnified the OZ-triggered O-2(-)-production 3.3-fold at 1 mu M, but inhibited it completely at 5 mu M. We used these critical concentrations to study the effects of OA on changes in phosphorylation and intracellular localization of cofilin. The OZ-induced dephosphorylation of cofilin was inhibited by 5 mu M OA but not by 1 mu M OA, Subcellular fractionation and immunoblotting revealed that 1 mu M OA increased cofilin on the phagosomal membranous fraction but 5 mu M OA decreased it. At 1 mu M, OA increased translocation of p47phox to membranes, which may explain in part the enhancing effect of 1 mu M OA. Confocal laser scanning microscopy showed that: (i) Cofilin diffused throughout the cytosol of resting cells, but accumulated at the plasma membranes forming phagocytic vesicles in activated cells. (ii) At 1 mu M, OA had little effect on the OZ-evoked translocation of cofilin, whereas 5 mu M OA suppressed it completely. (iii) OA alone, which could not trigger the phagocytic respiratory burst, did not cause any change in the distribution of cofilin at such concentrations. Furthermore, in a superoxide-producing cell-free system employing membranous and cytosolic fractions, affinity-purified anti-cofilin antibody showed an enhancing effect, These results suggest that cofilin participates in the superoxide production of the OZ-activated phagocytes through dephosphorylation and translocation, The roles of cofilin in the activated leukocytes will be discussed.